1. Field of the Invention
The present invention relates generally to the field of molecular biology and to its application in the utilization of nucleic acid transcripts which import biological molecules from the cell cytoplasm into mitochondria. The invention also concerns vectors incorporating DNA encoding the transcripts and various peptide/RNA mitochondrial import complexes. The nucleic acid segments copy RNA molecules which can transport additional RNA sequences, peptides or DNA molecules into the mitochondrial matrix. Also disclosed are methods of gene therapy directed at mitochondrial gene defects.
2. Description of Related Art
Mitochondria have been recognized as having a role in certain diseases and may be directly related to some aspects of the ageing process. An increased understanding of the function of mitochondrial genes in relation to the nuclear genes has therefore been the subject of much interest and research.
Current views of mitochondria hold that mitochondria import most small molecules and proteins from the cytoplasm. There is some evidence that RNA may also be imported, although the mechanism is not clear (Vestweber and Schatz, 1989).
The mammalian mitochondrial genome is different from nuclear genes in both structure and function. The human mitochondrial genome is small and economically packaged, lacking the large intervening sequences of non-coding DNA (introns) that constitute most of the nuclear genome. Mitochondrial amino acids are determined by a different genetic code than those in nuclear genes. Numerous other differences exist between nuclear and mitochondrial genes; for example, the monocistronic transcription from individual promoters and the extensive post transcriptional cleavage and ligation steps. Characteristic of mRNA synthesis directed by nuclear genes mitochondrial DNA is transcribed as a single polycistronic message subsequently cleaved to produce individual transfer, ribosomal and messenger RNA transcripts.
Mitochondrial biogenesis requires the participation of two distinct genetic compartments: the nuclear genome that contributes the vast majority of mitochondrial proteins and the mitochondrial genome that contributes 13 protein subunits to inter membrane enzymes of the respiratory chain (Anderson et al., 1981; Bibb et al., 1981). With the exception of two ribosomal RNA subunits and a complete set of tRNA species, the gene products necessary for replication transcription and translation of mitochondrial genes in cells of higher eukaryotes are derived entirely from the nucleus (Kruse et al., 1989; Parisi and Clayton, 1991; Attardi and Schatz, 1988). The set of nuclear genes required for replication and expression of the mitochondrial genome appears to include not only protein coding genes but loci that encode small RNA transcripts. Nuclear-encoded tRNAs have been observed in mitochondria from lower eukaryotes (Mottran et al., 1991; Nagley, 1989) and mitochondrial RNaseP in mammalian cells may include a nuclear encoded RNA subunit (Doersen et al., 1985). Even more definitive evidence for the participation of small RNA transcripts of nuclear origin in essential mitochondrial functions lies in the discovery of a mammalian RNase MRP that is presumed to generate primers for mitochondrial DNA replication (Chang and Clayton, 1987a; 1987b). This enzyme requires an RNA subunit (MRP-RNA) encoded by a single copy nuclear gene that is highly conserved among the mammalian species (Chang and Clayton, 1989; Yuan et al., 1989; Gold et al., 1989).
Most of the total cellular pool of MRP-RNA is localized within nucleoli (Reimer et al., 1988) but a small fraction of MRP-RNA partitions to mitochondria (Chang and Clayton, 1987b). The nuclear and cytoplasmic enzymes associated with MRP RNA exhibit some distinctions in RNase activity (Karwan et al., 1991). The mitochondrial RNase MRP cleaves a mitochondrial RNA substrate (transcribed in vitro) at a unique site between conserved sequence blocks (CSB) II and III. Mutations in CSB II and III severely inhibit the cleavage (Bennett and Clayton, 1990). In contrast, nuclear RNase MRP cleaves the same RNA substrate in multiple sites. Variations in the enzymatic properties of mitochondrial and nuclear RNases that contain identical MRP-RNA subunits are attributable to differences in apoprotein components (Karwan et al., 1991).
The mitochondrial inner membrane is impermeable to charged molecules so that transport of metabolites and proteins is accomplished by specialized carriers for small molecules (Aquila et al., 1987) and by an elaborate import apparatus for proteins (Sollner et al., 1991; Manning-Krieg et al., 1991). The partitioning of MRP-RNA to the mitochondrial compartment after transcription within the nucleus suggests the existence of a pathway by which RNA transcripts exit the nucleus and are imported across both the outer and inner mitochondrial membranes to the site of holoenzyme assembly within the mitochondrial matrix. However, such an import pathway has not been elucidated, nor has there been identification of sequence-specific targeting that might provide a signal for mitochondrial import.
The variable proportion of mutant mitochondrial genomes per cell results in cells with a range of bioenergetic capacities. Moreover, the expression of the whole genome is essential for the maintenance of mitochondrial bioenergetic function. Despite this knowledge of mitochondrial gene structure and of the biochemical steps involved in mitochondrial gene expression, relatively little is known about processes that regulate the expression of mammalian mitochondrial genes.
Mitochondrial function has been the subject of numerous studies, both in energy regulation and as a source of DNA mutations that may contribute to aging and degenerative diseases. Age dependent increases in deleted mitochondrial DNA, for example, have been found in the human heart (Hattori et al., 1991). Certain diseases such as Parkinson's disease, appear to be closely related to aging. Deletions in aging heart tissue are similar to those found in some Parkinson's patients and it has been speculated that some factors that accelerate mitochondrial DNA mutations may contribute to both Parkinson's disease and cardiomyopathy.
Other diseases and conditions may also be associated with defects in mitochondrial DNA. These include Kearns-Sayre syndrome and retinitis pigmentosa, ataxia, seizures, dementia and proximal muscle weakness (Grossman, 1990). A single base change in human mitochondrial DNA has been correlated with the appearance of Leber Hereditary Optic Neuropathy (LHON). LHON is a form of central optic nerve death resulting in blindness in affected individuals at a relatively early age, typically in their early twenties.
The identification of a region of RNA that may serve as a mitochondrial targeting signal has potential clinical significance. Several maternally inherited human diseases are associated with deletions and point mutations in the mitochondrial genome (Holt et al., 1988; Wallace et al., 1988; Shoffner et al., 1990; Goto et al., 1990). For example, myoclonic epilepsy and ragged-red fiber disease (MERRF) and mitochondrial myopathy, encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) are attributable to single base substitutions in tRNA.sup.Lys and tRNA.sup.Leu, respectively (Shoffner et al., 1990; Goto et al., 1990). The tRNA.sup.Lys mutation causes a general reduction in mitochondrial protein synthesis (Chomyn et al., 1991).
Prospects for gene therapy directed at mitochondrial gene defects are limited currently by the absence of methods for efficient introduction of foreign genetic material into mitochondria (discussed by Lander and Lodish, 1990). Mitochondrial dysfunction in cells of MERRF and MELAS patients may be correctable by linkage of a mitochondrial import signal to mitochondrial tRNA sequences expressed from nuclear trans-genes, without a requirement for direct genetic transformation of mitochondria.